The use of glycosaminoglycans, such as heparin, in various anticoagulant and antithrombotic therapies is well known. Sulodexide is a glycosaminoglycan of natural origin extracted from mammalian intestinal mucosa that posses an anticoagulant activity. Sulodexide has a sulfation degree lower than those of heparin, as shown by Radhakrishnamurthy B. et al., Atherosclerosis, 31, 217-229 (1978). The preparation of Sulodexide is described in U.S. Pat. No. 3,936,351, (incorporated herein by reference in its entirety).
Sulodexide is marketed in Europe under the trademark VESSEL DUE F(R) and is prescribed for the treatment of vascular pathologies with thrombotic risk such as peripheral occlusive arterial disease (POAD), healing of venous leg ulcers and intermittent claudication (See Harenberg J, Med. Res. Rev. vol. 18,1-20 (1998), Crepaldi G. et al., Atherosclerosis, 81, 233, (1990)), cardiovasculopathies, as described by Tramarin R. et al. Medical Praxis, 8, 1, (1987), cerebrovasculopathies as described by Sozzi C., Eur. Rev. Med. Pharmacol Sci. 6, 295, (1984) and venous pathologies of the lower limbs, as described by Cospite M. et al, Acta Therapeutica, 18, 149, (1992).
Diabetic nephropathy is a common and serious complication of the disease Diabetes Mellitus and is the leading cause of chronic renal failure (CRF) in the U.S., being responsible for one third of all CFR cases. The precise cause of the changes produced by diabetes in the kidney are not all known. However, it is known that a hallmark of diabetic nephropathy is the presence of protein in the urine (proteinuria) of patients. The protein albumin, which is normally present in plasma, is excreted in the urine of patients with diabetic nephropathy. This is referred to as albuminuria. The rate of excretion of this protein is a good indicator of the extent of the renal pathology. High levels of albumin excretion strongly predict accelerated diminution in glomerular filtration rate (GFR) and eventual renal failure. The degree of albumin excretion is used to divide patients into two groups; those with microalbuminuria (excretion of 200 mcg/min or less), and those with macroalbuminuria (excretion of more than 200 mcg/min).
The antihypertension drugs known as ACE inhibitors (ACEI) decrease albuminuria in diabetic nephropathy but the pathological changes in the kidney and renal functional deterioration may continue toward end stage renal disease (ESRD). ACEI are also less effective in type 2 or non-insulin dependent diabetic (DM2) nephropathy as compared to that seen in type 1 or insulin dependent diabetes (DM1). Salvetti A. et al. Drugs 57(5):665-693 (1999).
Diabetic nephropathy is a clinically well defined pathological condition characterized by proteinuria, hypertension, edema and renal insufficiency and generally occurs in patients suffering from diabetes for more than ten years. Diabetic nephropathy causes a number of structural changes in the kidney, the most characteristic of which is the glomerular injury detectable by the enlargement of the mesangium and by the thickening of the basement membrane in the glomerulus. (See Cecil Textbook of Medicine (pages 1281-1283), Edited by Goldman L. and Claude Bennett J., 21st Edition (2000), W. B. Saunders, Philadelphia).
Glycosaminoglycans such as Sulodexide are known to decrease albumin excretion in patients with diabetic nephropathy. The precise mechanism is not known but may include the following:                1) Restoration of the physiologic glomerular membrane anionic charge barrier via enhanced synthesis and sulfation of heparan sulfate in renal vascular membranes, and direct replenishment of renal heparan sulfate,        2) Inhibition of Transforming Growth Factor beta-1 (TGF beta-1) mediated mesangial matrix overproduction,        3) Inhibition of endothelin mediated tubulo-interstitial fibrosis, and        4) Inhibition of mesangial cell hyperplasia.(See Harenberg J., Med. Res. Rev. vol. 18, 1-20 (1998), Gambaro G. and Van Der Woude, J. Am. Soc. Nephrol. 11:359-368 (2000)).        
Kanwar Y. S. et al., Sem. Nephrol., 5, 307, (1985) and Groggel G. C. et al., Kidney Int., 33, 517, (1988), have produced evidence of the probable role of glycosaminoglycans in helping the integrity and the functioning of the renal cells.
Moreover, Canfield J. P. et al., Lab. Invest., 39, 505, (1978), previously showed a decrease of glycosaminoglycans in the glomerular basement membrane in many conditions of nephropathy, while Baggio B. et al., Nephron., 43, 187, (1986) showed an increased urinary elimination of glycosaminoglycans in diabetic, non-albuminuric, patients. This increased excretion of glycosaminoglycans in diabetic nephropathies was shown also by Partasarathy N. et al., Diabetes, 31, 738, (1982).
In addition, Diamond J. R. et al., Renal Physiol., 9, 366, (1986) and Parkerson M. B. et al., J. Clin. Invest., 81, 69, (1988), showed in animals the potential protective effect of heparin and its derivatives in models of experimental nephropathy not related to diabetic nephropathy, like chronic nephrosis from aminoglycosides and renal pathologies resulting from the subtotal renal ablation in the rat.
The use of heparin, low molecular weight heparin fractions, chemically modified heparins or low molecular weight dermatan sulfate in the treatment of both diabetic nephropathy and neuropathy has been investigated in the European Patent Publication EP 0513513 and U.S. Pat. No. 5,236,910 (incorporated herein by reference in its entirety). The possibility of therapeutic use was shown by means of pharmacological tests in animals: diabetes was caused by streptozotocin in Sprague Dawley male albino rats and the diabetic rats were treated with the above mentioned glycosaminoglycans. The effectiveness of treatment in animals was determined on the basis of the diminution of albuminuria and of the reduction of the thickness of the basal glomerular membrane and the increase of the glomerular anionic charges.
The use of Sulodexide to treat diabetic nephropathy is described in U.S. Pat. No. 5,496,807 (incorporated herein by reference in its entirety). This patent describes the effectiveness of Sulodexide in two clinical studies in which diabetic patients, some with microalbuminuria and some with macroalbuminuria, were given doses of Sulodexide. In one study Sulodexide was administered at the then recognized safe and effective dose of 1000 lipoproteinlipase releasing units/day (LRU's/day) (equivalent to 100 mg/day). This daily dose required two 250 LRU capsules of VESSEL DUE F(R) administrated twice a day. After 60 days, 8 out of 10 diabetic patients, 4 with microalbuminuria and 4 with macroalbuminuria showed an average decrease of 44% and 35% in albumin excretion respectively. Two of the patients with microalbuminia did not get any improvement. In addition, no substantial change in the glomerular filtration rate of any of the patients was noted at follow-up four months after the end of treatment.
The second clinical trial described in U.S. Pat. No. 5,496,807 involved three microalbuminuric and two macroalbuminuric diabetic patients administered 600 LRU/day of Sulodexide by intramuscular route for 21 days. All five of these patients showed a significant lowering of albumin excretion.
In neither of these studies, disclosed in the '807 patent, was the improvement in albumin excretion shown to be maintained after the Sulodexide administration ended. The dose range disclosed in this patent specification and the claims was 500 to 1500 L.R.U.'s or 50 to 150 mg./day.
Recently, the use of glycosaminoglycans, including Sulodexide, in the treatment of diabetic nephropathy has been reviewed by Gambaro G. and Van Der Woude in J. Am. Soc. Nephrol. 11: 359-368 (2000). In this review Sulodexide is described as being active in preventing diabetic nephropathy in an experimental animal model, Gambaro et al. Kidney Int. 42: 285-291, 1992. In addition, Sulodexide is said to have been shown to reduce albuminuria in both insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM). (See Skrha J. et al. Diabetes Res. Clin. Pract. 38: 25-51 (1997), Solini A. et al. Diabetes Nutr. Metab. 7: 304-307, (1994), Poplawska A. et al. Diabetes Res Clin. Pract. 38: 109-114, (1997), Dedov. I. et al. Nephrol Dial Transplant 12:2295-2300, (1997) and Szelanowska M. et al. Curr Med. Res. Opin. 13:539-545, (1997)).
In these studies Sulodexide treatment in IDDM was found to be consistently effective in reducing microalbuminuria. However this hypoalbuminuric effect was observed in only 30 to 50% of NIDDM patient, and at most the effect lasted only several weeks after the withdrawal of the Sulodexide. In the studies reviewed by Gambaro the maximum dose of Sulodexide used was 100 mg/day orally and more typically doses of only 60 mg/day were used either by the oral route or by intra muscular injection. Previous experience in using Sulodexide to treat other pathological conditions had shown that doses of 25-100 mg/day (250-1000 LRU's) were considered clinically effective.
In addition, another reason for the use of such low doses was the concern over possible risks and side effects of a drug such as Sulodexide. For example Sulodexide is known to have antithrombotic activity equal to that of heparin, Thomas D. P. et al. Ann. N.Y. Acad. Sci., 556, 313 (1989) and to completely prevent clot formation at high doses. In primates the oral administration of 10 mg/kg of Sulodexide increased tissue plasminogen activator (TPA) from 5 to 10 ng/ml and produced an increase of U-PA from 3 to 6.5 ng/ml. This suggests that Sulodexide is a strong anticoagulant, antithrombotic and profibromolytic agent. Callas D. D. et al., Thromb. Hemost., 19 (Suppl. 1), 49 (1993).
Effective oral doses of Sulodexide have been in the range of 25-100 mg/day or 250-1000 LRU/day for the treatment of vascular pathologies Harenberg J., Med. Res. Rev. vol. 18, 1-20 (1998). The largest dose reported in the literature to our knowledge for the treatment of diabetic nephropathy was an oral dose of 1000 LRU/day or 100 mg/day. This dose did decrease the rate of albumin excretion in patients with NIDDM (type II diabetes or DM2) but the albumin excretion rate was found again increased four months after cessation of treatment Solina A. Diab. Nutr. Metabo., 7, 304 (1994).
The art concerning the use of Sulodexide to treat vascular conditions teaches that doses of 100 mg/day or 1000 LRU's or less are effective and that higher doses may produce unwanted or dangerous heparin like effects. However, doses in the range of 100 mg./day (1000 LRU's) or less, have not proved effective in all subjects with diabetic nephropathy and have not been shown to produce lasting improvement in the albumin excretion rate in treated patients. (See Harenberg J. Med. Res. Rev. vol. 18, 1-20 (1998) and Gambaro G. and Van Der Woude, J. Am. Soc. Nephrol. 11:359-368 (2000)). Furthermore, oral administration is much more desirable and less problematic to the patient as compared to intramucular administration.
For these reasons there is a need for oral formulations of Sulodexide in dosage formulations adapted to adequately treat diabetic nephropathy in both macro and micro albuminic patients and to produce lasting improvement in albumin excretion rate in diabetic patients.